Interstitial cells in the primate gastrointestinal tract

Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine⁺ mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.     

Immunomagnetic enrichment of interstitial cells of Cajal

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was approximately 63-fold and approximately 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca(2+) or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.     

Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ

Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca²⁺ imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca²⁺ transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca²⁺ transient wave fronts propagated through the ICC-MY networks at 2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca²⁺ transients in LM but had no visible effect on the transients in ICC-MY. -Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca²⁺ entry (Ni²⁺ and mibefradil) and elevated external K⁺ reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca²⁺ transients in ICC-MY. These data show that global Ca²⁺ transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca²⁺ handling mechanisms and entrained by voltage-dependent Ca²⁺ entry and coupling of cells via gap junctions. Ca²⁺ signaling; slow waves; gastrointestinal motility     

Relationships between neurokinin receptor-expressing interstitial cells of Cajal and tachykininergic nerves in the gut

The so-called interstitial cells of Cajal (ICC) are distributed throughout the muscle coat of the alimentary tract with characteristic intramural location and species-variations in structure and staining. Several ICC sub-types have been identified: ICC-DMP, ICC-MP, ICC-IM, ICC-SM. Gut motility is regulated by ICC and each sub-type is responsible for the electrical activities typical of each gut region and/or muscle layer. The interstitial position of the ICC between nerve endings and smooth muscle cells has been extensively considered. Some of these nerve endings contain tachykinins. Three distinct tachykinin receptors (NK1r, NK2r and NK3r) have been demonstrated by molecular biology. Each of them binds with different affinities to a series of tachykinins (SP, NKA and NKB). In the ileum, SP-immunoreactive (SP-IR) nerve fibers form a rich plexus at the deep muscular plexus (DMP), distributed around SP-negative cells, and ICC-DMP intensely express the SP-preferred receptor NK1r; conversely a faint NK1r-IR is detected on the ICC-MP and mainly after receptor internalization was induced by agonists. ICC-IM are never stained in laboratory mammals, while those of the human antrum are NK1r- IR. RT-PCR conducted on isolated ileal ICC-MP and gastric ICC-IM showed that these cells express NK1r and NK3r. Colonic ICC, except those in humans, do not express NK1r-IR, at least in resting conditions. Outside the gut, NK1r-IR cells were seen in the arterial wall and exocrine pancreas. In the mouse gut only, NK1r-IR is present in non-neuronal cells located within the intestinal villi, so-called myoid cells, which are c-kit-negative and alpha-smooth muscle actin-positive. Immunohistochemistry and functional studies confirmed that ICC receive input from SP-IR terminals, with differences between ICC sub-types. In the rat, very early after birth, NK1r is expressed by the ICC-DMP and SP by the related nerve varicosities. Studies on pathological conditions are few and those on mutant strains practically absent. It has only been reported that in the inflamed ileum of rats the NK1r-IR ICC-DMP disappear and that at the peak of inflammatory conditions ICC-MP are NK1r-IR. In the ileum of mice with a mutation in the W locus, ICC-DMP were seen to express c-kit-IR but not NK1-IR, and SP-IR innervation seems unchanged. In summary, there are distinct ICC populations, each of them under a different tachykininergic control and, likely, having different functions. Further studies are recommended at the aim of understanding ICC involvement in modulating/transmitting tachykininergic inputs.     

Morphology of the interstitial cells of Cajal of the human ileum from foetal to neonatal life

The so-called interstitial cells of Cajal myenteric plexus (ICC-MP), interstitial cells of Cajal intramuscular (ICC-IM) and interstitial cells of Cajal deep muscular plexus (ICC-DMP) are the three types of ICC endowed within the intestinal muscle coat where they play different roles in gut motility. Studies on ICC ontogenesis showed ICC-MP in the human ileum by 7-9 weeks while information on ICC-IM and ICC-DMP in foetuses and newborns are not exhaustive. Functional recordings in the fasting state of prematurely born babies aged 28-37 weeks showed immature ileal motility. To gain more information on the time of appearance of the three ICC types in the human ileum and on the steps of the acquisition of mature features, we studied by c-kit immuno-histochemistry foetuses aged 17-27 weeks and newborns aged 36-41 weeks. In parallel, the maturative steps of enteric plexuses and muscle layers were immunohistochemically examined by using anti-neuron specific enolase (NSE), anti-S-100 and anti-alpha smooth muscle actin (alphaSMA) antibodies. The appearance and differentiation of all the ICC types were seen to occur in concomitance with those of the related nerve plexuses and muscle layers. ICC-MP appeared first, ICC-IM and ICC-DMP later and their differentiation was incomplete at birth. In conclusion, the ICC-MP, the intestinal pacemaker cells, in spite of absence of food intake, are already present during the foetal life and the ICC-IM appear by pre-term life, thus ensuring neurotransmission. The ICC-DMP and their related nerve plexus and smooth muscle cells, i.e. the intestinal stretch receptor, begin to differentiate at birth. These findings might help in predicting neonatal ileal motor behaviour and in interpreting the role of ICC abnormalities in the pathophysiology of intestinal motile disorders of neonates and young children.     

P2Y11 receptors activate adenylyl cyclase and contribute to nucleotide-promoted cAMP formation in MDCK-D(1) cells. A mechanism for nucleotide-mediated autocrine-paracrine regulation.

Extracellular nucleotides activate P2Y receptors, thereby increasing cAMP formation in Madin-Darby canine kidney (MDCK-D(1)) cells, which express P2Y(1), P2Y(2), and P2Y(11) receptors (Post, S. R., Rump, L. C., Zambon, A., Hughes, R. J., Buda, M. D., Jacobson, J. P., Kao, C. C., and Insel, P. A. (1998) J. Biol. Chem. 273, 23093-23097). The cyclooxygenase inhibitor indomethacin (indo) eliminates UTP-promoted cAMP formation (i.e. via P2Y(2) receptors) but only partially blocks ATP-promoted cAMP formation. The latter response is completely blocked by the nonselective P2Y receptor antagonist suramin. We have sought to identify the mechanism for this P2Y receptor-mediated, indo-resistant cAMP formation. The agonist rank order potencies for cAMP formation were: ADP beta S > or = MT-ADP > 2-MT-ATP > ADP, ATP, ATP gamma S > UTP, AMP, adenosine. We found a similar rank order in MDCK-D(1) cells overexpressing cloned green fluorescent protein-tagged P2Y(11) receptors, but the potency of the agonists was enhanced, consistent with a P2Y(11) receptor-mediated effect. cAMP generation by the P2Y(1) and P2Y(11) receptor agonist ADP beta S was not inhibited by several P2Y(1)-selective antagonists (PPADS, A2P5P, and MRS 2179). Forskolin synergistically enhanced cAMP generation in response to ADP beta S or PGE(2), implying that, like PGE(2), ADP beta S activates adenylyl cyclase via G(s), a conclusion supported by results showing ADP beta S and MT-ADP promoted activation of adenylyl cyclase activity in MDCK-D(1) membranes. We conclude that nucleotide-promoted, indo-resistant cAMP formation in MDCK-D(1) cells occurs via G(s)-linked P2Y(11) receptors. These data describing adenylyl cyclase activity via endogenous P2Y(11) receptors define a mechanism by which released nucleotides can increase cAMP in MDCK-D(1) and other P2Y(11)-containing cells.     

Similar Rates of Phosphatidylinositol Hydrolysis Following Activation of Wild-Type and Truncated Rat Neurokinin-1 Receptors

Abstract: The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC₅₀ of substance P-induced accumulation of ³H-inositol phosphates. Substance P-induced responses continued for 30–45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of ³H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.     

Activation of neurokinin 1 receptors on interstitial cells of Cajal of the guinea-pig small intestine by substance P

 The aims of this work were to determine whether cells that are similar to the interstitial cells of Cajal (ICC) and have immunoreactivity for the neurokinin 1 (NK1) receptor are indeed ICC; to determine whether the agonist, substance P, binds to and activates the receptor on presumptive ICC; and to investigate the relationship between substance P-immunoreactive nerve fibres and ICC. ICC at the level of the myenteric plexus and in the deep muscular plexus in the duodenum and ileum of the guinea-pig were investigated. Immunoreactivities for the ICC marker, Kit, and the NK1 receptor were colocalised in ICC of the myenteric and deep muscular plexuses. In tissue fixed immediately after its removal from the animal, NK1 receptor-immunoreactive ICC were found at the level of the myenteric plexus in the duodenum, but not in the ileum, and in the deep muscular plexus in the duodenum and ileum. The majority of receptor immunoreactivity was on the cell surface. ICC were exposed to substance P (10^–7 M), initially at 4°C for 1 h to allow the agonist to bind, followed by incubation at 37°C to allow receptor internalisation to proceed. Exposure to substance P caused the NK1 receptor immunoreactivity to aggregate in clumps in the cytoplasm of ICC of the myenteric and deep muscular plexuses, including the ICC of the myenteric plexus of the ileum, where NK1 receptor immunoreactivity was not seen if tissue was not exposed to substance P. Substance P, to which the fluorescent label, cyanine 3.18 (Cy-3), was coupled, bound to the ICC. The Cy-3-substance P was internalised with the receptor following warming to 37°C. Many, but not all, ICC were closely apposed by nerve fibres with immunoreactivity for substance P. It is concluded that the NK1 receptor immunoreactivity on ICC represents receptor that is functional in the sense that it binds the natural agonist substance P and undergoes agonist-induced internalisation. ICC are likely to receive excitatory innervation from the close approaches of tachykinin-containing nerve fibres. Accepted: 10 March 1998     

Expression and Significance of Neuroligins in Myenteric Cells of Cajal in Hirschsprung's Disease

Purpose

The aim of this study was to investigate the expression and significance of neuroligins in myenteric cells of Cajal (ICC-MY) in Hirschsprung’s disease (HSCR).

Methods

Longitudinal muscle with adherent myenteric plexus (LMMP) from surgical excision waste colon of HSCR children were prepared by peeling off the mucous layer, sub-mucosal layer and circular muscle. Neuroligins, c-Kit (c-Kit-immunoreactivity representing ICC) and their relationship were assessed by double labeling immunofluorescence staining. ICC-MY were dissociated and cultured from LMMP by enzymolysis method, and were purified and analyzed using a combination of magnetic-activated cell sorting (MACS) and flow cytometry (FCM). Western-blot analysis was applied to compare and evaluate the expression levels of neuroligins in ICC-MY which were dissociated from different segments of HSCR (ganglionic colonic segment, transitional colonic segment and aganglionic colonic segment).

Results

Neuroligins and c-Kit were expressed on the same cells (ICC-MY); ICC-MY were dissociated, cultured and purified. For HSCR, neuroligins were expressed significantly in ICC-MY from ganglionic colonic segments, moderately in those from transitional colonic segments and down-regulated significantly in those from aganglionic colonic segments.

Conclusions

Neuroligins were expressed in ICC-MY of human beings, and the expression varies from different segments of HSCR. This abnormal expression might play an important role in the pathogenesis of this disease through affecting the synaptic function of ICC-MY.     

Interstitial cell network volume is reduced in the terminal bowel of ageing mice

Ageing is associated with impaired neuromuscular function of the terminal gastrointestinal (GI) tract, which can result in chronic constipation, faecal impaction and incontinence. Interstitial cells of cajal (ICC) play an important role in regulation of intestinal smooth muscle contraction. However, changes in ICC volume with age in the terminal GI tract (the anal canal including the anal sphincter region and rectum) have not been studied. Here, the distribution, morphology and network volume of ICC in the terminal GI tract of 3‐ to 4‐month‐old and 26‐ to 28‐month‐old C57BL/6 mice were investigated. ICC were identified by immunofluorescence labelling of wholemount preparations with an antibody against c‐Kit. ICC network volume was measured by software‐based 3D volume rendering of confocal Z stacks. A significant reduction in ICC network volume per unit volume of muscle was measured in aged animals. No age‐associated change in ICC morphology was detected. The thickness of the circular muscle layer of the anal sphincter region and rectum increased with age, while that in the distal colon decreased. These results suggest that ageing is associated with a reduction in the network volume of ICC in the terminal GI tract, which may influence the normal function of these regions.     

Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers, and mediators of neurotransmission in the gastrointestinal tract.

BACKGROUND: Interstitial cells of Cajal (ICCs) are mesenchymal cells that play critical roles in gastrointestinal motility as electrical pacemakers and mediators of neuromuscular neurotransmission. Although depletions of ICCs have been implicated in several gastrointestinal motor disorders, quantification of these cells has been difficult due to their varied morphology, regionally changing network density, and overall scarcity. Our goal was to evaluate flow cytometry (FCM) for the enumeration of ICCs. METHODS: We identified murine ICCs in live gastrointestinal muscles or primary cell cultures grown in the presence or absence of stem cell factor (SCF)-expressing STO fibroblasts with fluorescent Kit (CD117) antibodies. Because this technique also labels resident macrophages nonspecifically, we identified the latter with additional fluorescent antibodies. Dispersed cells were analyzed by FCM. RESULTS: ICCs represented 1.63 +/- 0.17% of the total cell count in the distal stomach (n = 18 mice) and 5.85 +/- 0.84% in the proximal colon and 6.28 +/- 0.61% in the distal colon (n = 3 mice). In fundic muscles of W/WV mice (n = 5) that virtually lack ICCs, very few Kit+ cells were detected. FCM identified approximately 2.6- to 7.3-fold more Kit+ ICCs in small intestinal cell cultures grown on STO fibroblasts expressing membrane-bound SCF (n = 6) than in cultures stimulated with soluble SCF (n = 6). CONCLUSIONS: FCM is a sensitive and specific method for the unbiased quantification of ICCs.     

Ether-a-go-go-related gene 3 is the main candidate for the E-4031-sensitive potassium current in the pacemaker interstitial cells of Cajal

The interstitial cells of Cajal (ICC), as pacemaker cells of the gut, contribute to rhythmic peristalsis and muscle excitability through initiation of slow-wave activity, which subsequently actively propagates into the musculature. An E-4031-sensitive K(+) current makes a critical contribution to membrane potential in ICC. This study provides novel features of this current in ICC in physiological solutions and seeks to identify the channel isoform. In situ hybridization and Kit immunohistochemistry were combined to assess ether-a-go-go-related gene (ERG) mRNA expression in ICC in mouse jejunum, while the translated message was assessed by immunofluorescence colocalization of ERG and Kit proteins. E-4031-sensitive currents in cultured ICC were studied by the whole cell patch-clamp method, with physiological K(+) concentration in the bath and the pipette. In situ hybridization combined with Kit immunohistochemistry detected m-erg1 and m-erg3, but not m-erg2, mRNA in ICC. ERG3 protein was colocalized with Kit-immunoreactive ICC in jejunum sections, but ERG1 protein was visualized only in the smooth muscle cells. At physiological K(+) concentration, the E-4031-sensitive outward current in ICC was different from its counterpart in cardiac and gut smooth muscle cells. In particular, inactivation upon depolarization and recovery from inactivation by hyperpolarization were modest in ICC. In summary, the E-4031-sensitive currents influence the kinetics of the pacemaker activity in ICC and contribute to maintenance of the resting membrane potential in smooth muscle cells, which together constitute a marked effect on tissue excitability. Whereas this current is mediated by ERG1 in smooth muscle, it is primarily mediated by ERG3 in ICC.     

Purification of interstitial cells of Cajal by fluorescence-activated cell sorting

Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic because of the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Because this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000–40,000 cells and 60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched 6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested 2,000–4,000 cells from single gastric corpus-antrum muscles and detected an 30-fold increase in c-kit mRNA, no enrichment of mast cells, and an 4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses. mouse; c-kit; macrophage; dendritic cell; mast cell     

Substance P (NK1) receptor in relation to substance P innervation in rat duodenum after irradiation

It has previously been shown that high dose of irradiation to the rat abdomen leads to an increased level of substance P (SP) in the duodenum. In the present study the pattern of distribution of NK1 receptors (NK1-R) in rat duodenum after irradiation (5-30 Gy), was examined at the same time-point (7 days) after irradiation, comparisons being made with the distribution of SP-innervation. Immunohistochemical methods were used. In controls, NK1-R-like immunoreactivity (-LI) was detected in epithelial cells, in cells in the region of the intestinal cells of Cajal within the deep muscular plexus (ICC-DMP), in neuronal cells in the myenteric plexus, and variably in granulocytes in the mucosa. Irradiation with 5-10 Gy did not lead to obvious changes in the pattern of NK1-R-LI. After irradiation with the highest doses (25-30 Gy), the mucosa was often gravely damaged, displaying granulation tissue. No epithelial NK1-R-LI was detected in this tissue, but was present in less affected mucosa after these doses. In the region of the ICC-DMP, in the myenteric plexus, and in granulocytes, NK1-R-LI was detected also after high dose irradiation. However, the degree of NK1-R-LI in the region of the ICC-DMP was somewhat lower than seen in controls and after low doses. SP-immunoreactive nerve fibers were present in the regions where NK1-R-LI was detected. These findings support a suggestion that an increased level of SP after irradiation may contribute to the dose-dependent gastrointestinal adverse effects that occur after radiotherapy.     

Kit K641E oncogene up-regulates Sprouty homolog 4 and Trophoblast glycoprotein in interstitial cells of Cajal in a murine model of gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the receptor tyrosine kinase KIT are present in most GIST. KIT K642E was originally identified in sporadic GIST and later found in the germ line of a familial GIST cohort. A mouse model harbouring a germline Kit K641E mutant was created to model familial GIST. The expression profile was investigated in the gastric antrum of the Kit ^K641E murine GIST model by microarray, quantitative PCR and immunofluorescence. Gja1/Cx43 , Gpc6 , Gpr133 , Pacrg , Pde3a , Prkar2b , Prkcq/Pkce , Rasd2 , Spry4 and Tpbg/5T4 were found to be up-regulated. The proteins encoded by Gja1/Cx43 , Pde3a , Prkcq/Pkce were localized in Kit-ir ICC in wild-type and Kit ^K641E animals while Spry4 and Tpbg/5T4 were detected in Kit-ir cells only in Kit ^K641E, but not in Kit ^WT/WT animals. Most up-regulated genes in this mouse model belong to the gene expression profile of human GIST but also to the profile of normal Kit⁺ ICC in the mouse small intestine. Spry4 and Tpbg/5T4 may represent candidates for targeted therapeutic approaches in GIST with oncogenic KIT mutations.     

Natural killer cell functions mediated by the neuropeptide substance P

The neuropeptide substance P (SP) can modulate a number of immunological functions in vitro and in vivo. Here, we investigated if SP boosts migration and cytotoxicity of natural killer cells, thus providing a further link between "innate immunity" and neurogenic inflammatory processes like asthma bronchiale. We demonstrate a dose-dependent effect of SP on natural killer cell migration with a maximal response at 10(-8) M SP. SP was shown to stimulate unstimulated as well as interleukin-2 (IL-2)-activated natural killer cells. Stimulation of natural killer cell migration was neurokinin-1 receptor dependent. Furthermore, mRNA encoding the neurokinin-1 receptor was demonstrated as being present in natural killer cells using RT-PCR while mRNA of the neurokinin-2 receptor was not detectable. Additionally, SP seems to influence specific cytotoxicity against Raji and K567 effector cells by a receptor-independent mechanism. In conclusion, our data indicate that functionally active neurokinin-1 receptors can be expressed by human natural killer cells. Substance P might therefore be a novel link between neural structures and innate immunity.     

Substance P and Neurokinin 1 receptor - expression is affected in the ileum of mice with mutation in the W locus

The tachykinin substance P (SP) acts on the gut muscle coat via its preferred receptor, neurokinin 1 (NK1r). In the mouse ileum, NK1r-immunoreactivity (NK1r-IR) was detected in neurons, in the interstitial cells of Cajal at the deep muscular plexus (ICC-DMP) and the myoid cells of the villi. SP-IR was detected in neurons and varicose nerve fibers, which were especially numerous at the DMP and closely associated with the ICC-DMP. In mice with a mutation in the W locus (ckit mutant animals), innervation is suggested to be normal although few studies have actually tested this hypothesis. Indeed, studies demonstrating ICC-DMP integrity are lacking and whether SP- and NK1r-IR are normal in these animals has not been investigated. Our aim was to perform an immunohistochemical study on the ileum of a strain of heterozygous mice with a mutation in the W locus, the W(e/+) mice, to test this hypothesis. SP-IR nerve fibers were significantly more numerous than in wild type mice; NK1r-IR was clustered on the plasma membrane and also intracytoplasmatic in the neurons, but absent in the ICC-DMP. The richness in SP-IR nerve fibers and the NK1r-IR distribution in the neurons, similar to that of activated cells, might be attempts to compensate for the SP preferred receptor absence at the ICC-DMP. In conclusion, SP content and NK1r expression are noticeably different in c-kit mutants with respect to wild type mice, and probably causing an anomalous tachykininergic control of intestinal motility. Physiological studies on Wmutant mice have to take into account that innervation in this animal model is affected by the c-kit mutation.     

Modulation of basal and stress-induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L-822429

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self-regulating role of SP, we used a selective neurokinin-1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress-evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L-822429 represents a potent and selective rat NK1 receptor antagonist. Intra-amygdaloid administration of L-822429 via inverse microdialysis enhanced basal, but attenuated swim stress-induced SP release, while the low-affinity enantiomer of L-822429 had no effect. Using light and electron microscopy, synaptic contacts between SP-containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self-regulatory capacity of SP-mediated neurotransmission that differs in the effect on basal and stress-induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor-mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.